1. Field of the Invention
The present invention is directed to a method and apparatus for automated dispensing. More particularly, the present invention is directed to an automated dispensing system that dispenses a predetermined and precise amount of fluid into the wells of a multi-welled dish of the type usually employed for carrying out immunoassay and biochemical reactions.
2. Related Art
In many chemical and biochemical reactions and processes, it is necessary to distribute reagent or solution precisely and rapidly to multiple containers, One such process is the synthesis of oligonucleotides. Oligonucleotides play increasing and critical roles in diagnostic medicine, forensic medicine, and molecular biology research. In a conventional oligonucleotide synthesis process by phosphoramidite coupling, bases are sequentially coupled to a solid support. For example, the first nucleoside, protected at the 5' position, is derivatized to a solid support, usually controlled pore glass. The sugar group of the first nucleoside is deprotected or detritylated using an appropriate reagent to produce a colored product that may be monitored for reaction progress. The second nucleotide, that has the phosphorous, sugar and base groups protected, is added to the growing chain, usually in the presence of a tetrazole catalyst. The unreacted first nucleoside is capped to avoid perpetuating errors, using reagents such as acetic anhydride and N-methylimidazole. The phosphite triester is oxidized to form the more stable phosphate triester, usually using an iodine reagent. This process is repeated as needed to produce the desired length and sequence of the oligonucleotide. At the conclusion of the synthesis, the cleavage from the solid support is done, usually using aqueous ammonia. The oligonucleotides are then deprotected and dried to remove the ammonia solution. The oligonucleotides can then be resuspended in water, and quantitated using vertical spectrophotometry.
An apparatus and method for synthesizing an array of oligonucleotides is disclosed in U.S. Pat. Nos. 5,472,672 and 5,529,756. The foregoing two patents describe a synthesis apparatus that can be used to synthesize 96 oligonucleotides at one time using a standard microtiter well spacing format (8.times.12 array). The synthesis apparatus includes a delivery assembly for controlling delivery of the liquid reagents required in the synthesis process through an array of nozzles. A transport mechanism is provided so that the array of wells in the microtiter plate can be aligned with the array of nozzles in the delivery assembly. The delivery assembly is part of a head assembly that is coupled to a base assembly that includes the microtiter plate. A sliding seal is located between the bottom surface of the head assembly and the top surface of the base assembly to environmentally contain both the reactions wells and the nozzles in a common chamber. The sliding seal is used to exclude water and oxygen from the common reaction chamber during synthesis. Phosphoramidites are sensitive to hydrolysis by tracing of water, and to oxidation by contact with air. Because the coupling reactions are rapid and irreversible, it is necessary to exclude both water and oxygen from the reaction chamber during synthesis.
The apparatus described in U.S. Pat. Nos. 5,472,672 and 5,529,756 can be used to synthesize oligonucleotides in the reaction wells of a synthesis plate. Once synthesis is complete, the synthesis plate is removed from the apparatus and stacked on top of a second 96-well deprotection plate for the oligonucleotide cleavage step. The cleavage step requires pipetting 200 microliters of concentrated NH.sub.4 OH (ammonium hydroxide) into each well of the synthesis plate. The cleavage step is repeated twice with fresh aliquots of ammonia.
The cleavage step of pipetting concentrated NH.sub.4 OH into each well of the synthesis plate can be carried out by manually pipetting the appropriate aliquot into each well since the cleavage step does not require a controlled environment such as in the reaction chamber of the synthesis apparatus described above. Alternatively, the pipetting step could be carried out using an automated pipetting device, such as the automatic fluid dispenser for a multi-welled dish disclosed in U.S. Pat. No. 5,046,539. The fluid dispenser in this patent uses a single pipette. The multi-welled dish is moved using stepper motors in two directions to correspond to the two-dimensional array of rows and columns of the multi-welled dish. A stepper motor is also used to bias a plunger to force air, or other gas under pressure, into the single pipette. In order to deposit a precise amount of fluid in each well, the stepper motor is calibrated to control the amount of force exerted by the plunger.
The fluid dispenser disclosed in U.S. Pat. No. 5,046,539 has numerous drawbacks. Only a single pipette is used in the fluid dispenser to simplify sterilization procedures. However, because only a single pipette is used, the fluid dispenser must be configured for two-dimensional movement so that all of the wells in the multi-welled dish can be filled. The fluid dispenser fills the wells one at a time with the single pipette, offering little time savings over manually pipetting into the wells one at a time. Additionally, the fluid dispenser does not provide an accurate means for dispensing a calibrated amount of fluid. Controlling the force of the plunger by calibrating a stepper motor does not provide an accurate calibration, particularly for fluids of varying viscosity.
The fluid dispenser disclosed in U.S. Pat. No. 5,046,539 has a further drawback in that it cannot accurately dispense ammonium hydroxide. Ammonium hydroxide is very volatile at room temperature. Therefore, at room temperature, ammonium hydroxide expands continually. This results in continual loss of fluid from the end of the pipette.
A syringe pump is conventionally used to pump reagents in chemical and biochemical processes. However, syringe pumps are susceptible to leakage when volatile fluids are used. A volatile fluid will expand, causing a "blow-by" condition whereby the gas comes out of solution, and leaks out between the pump piston and the seal. Syringe pumps are typically configured with a long nozzle that terminates at a delivery end. When such a syringe pump is used to pump a volatile fluid such as NH.sub.4 OH, the volatile fluid will keep dribbling out of the delivery end. Consequently, a syringe pump must be configured with a valve at the delivery end of the nozzle in order to reliably and accurately dispense volatile fluids. To deliver a calibrated quantity of a volatile fluid with a conventional syringe pump, both the pump and a valve on the delivery end of the nozzle must be controlled.
Therefore, there is a need in the art for a device that can dispense a calibrated quantity of fluid simultaneously into a plurality of wells. Particularly, there is a need in the art for a device that can dispense a calibrated quantity of ammonium hydroxide simultaneously into a plurality of wells in a microtiter plate for oligonucleotide cleavage. There is a further need for a device that can dispense a calibrated quantity of volatile fluids, as well as fluids of varying viscosities.